Rapid and accurate detection of SARS-CoV-2 using the RHAM technology

Rapid and sensitive detection of pathogens is of utmost importance in interrupting the transmission chain of infectious diseases. In recent years, this has proven to be vital during the coronavirus disease (COVID-19) global pandemic that put countless lives at risk. Numerous molecular diagnostic methods were used, including RT-PCR, NASBA, E-SDA, E-RCA, LAMP, and RPA. However, these technologies potentially require primer optimization and complex instruments. Here, we propose the RHAM (RNase Hybridization-Assisted amplification) system as a rapid, specific, and sensitive molecular diagnosis platform. Combining the LAMP and RNase HII-mediated fluorescent reporter, the RHAM system can amplify and visualize the target in one isothermal system with high sensitivity (5 × 102 copies/mL). There was no cross-reactivity with other common respiratory viruses. Analysis of clinical samples revealed the RHAM system to generate positive signals within 15 min without false positive or negative results. The present study shows that RHAM is not only an ideal platform for detecting pathogens, such as SARS-CoV-2 but can be potentially applied in POCT settings.


Development of the RHAM system for detecting SARS-CoV-2 RNA
In the fluorescence-based LAMP detection protocol, reverse transcription of the target genome generates complementary DNA (cDNA).Meanwhile, in the single tube reaction, LAMP primers anneal to the cDNA and the cDNA is amplified using Bst DNA polymerase.The resulting product contained is a repeat of the amplicon linked by a single-stranded loop in a long concatemer (Fig. 1A,B) 16 .To enhance LAMP detection, RNase HII and the fluorescent probe were added to the LAMP reaction mix for the specific detection of amplicons targeting Orf1ab and N gene.During the LAMP process, the ribonucleotide-containing fluorescent probe, labeled with a fluorophore and a quencher, can hybridize with the single-stranded LAMP products.Upon recognition by RNase HII, the RNA-DNA hybrid was then cleaved, resulting in the release of the fluorophore (Fig. 1C).We combined the RNase HII, ribonucleotide-containing fluorescent probe with the LAMP system (hereafter named RHAM).
To compare LAMP and RHAM, experiments were performed for the detection of SARS-CoV-2.The realtime kinetics of positive (purified SARS-CoV-2 RNA reference, 500 copies/reaction) and negative samples (no template control) targeting the Orf1ab gene or N gene in LAMP reactions were monitored.Positive samples were found to display fluorescence after incubation for 15 min; however, both positive and negative samples showed similar fluorescence intensities after incubation for 27 min due to amplification of primer dimer (Supplementary Fig. 1), indicating the risk of a high false-positive rate, making it difficult to discriminate negative and low concentration positive result (Fig. 2A,B).To evaluate the performance of RNase HII, a positive fluorescent signal can be observed when adding both the ribonucleotide-containing fluorescent probe and the target single-stranded (ssDNA) compared to the negative control (without ssDNA; Fig. 2C,D).Next, the RHAM system results showed that the single pot system can robustly detect purified SARS-CoV-2 RNA targeting either Orf1ab or N gene in less than 10 min without a false positive signal in comparison to the LAMP system (Fig. 2E,F).Therefore, the RHAM system showed an evident improvement compared to LAMP.The above data indicate that time-to-positivity (TTP) value of RHAM is superior to or equal to LAMP and has stronger specificity compared with LAMP method for SARS-CoV-2 RNA detection.It is worth noting that the well-designed LAMP assays will not produce secondary structures and non-specific amplification products.We provide the RHAM assays as a choice for the primers which might generate non-specific amplification in the other assays, so that the available primers can be more easily selected.

RHAM is highly sensitive and specific
RHAM was established successfully for the rapid detection of SARS-CoV-2; however, its performance required further clarification.To assess the sensitivity and specificity of RHAM, its detection limit was examined using SARS-CoV-2 RNA samples.A series of diluted SARS-CoV-2 RNA samples (1 × 10 5 -5 × 10 2 copies/mL) were prepared, with each added to 25 μL of RHAM reaction mix targeting Orf1ab (Fig. 3A) or N gene (Fig. 3B).We then monitored the fluorescence of each dilution and calculated their coefficient of variations (CVs).All SARS-CoV-2 RNA positive samples showed fluorescence signals within 25 min.Both LAMP and RHAM amplified all the tested dilutions with similar TTP values.The no template control did not display any fluorescence signal in RHAM (Fig. 3A,B).However, using the same primer sets, LAMP may produce false positive signal in ~ 30 min (Fig. 3C,D).The TTP showed good correlation to the concentration of templates in the RHAM system (Fig. 3E,F).The specificity of the orf1ab and N gene primer sets and probe was also analyzed for SARS-CoV-2 diagnostics (Fig. 4A,B).Nucleic acids of other respiratory pathogens, including coronavirus (HKU1, OC43, NL63, 229E), SARS coronavirus, MERS coronavirus, influenza A H1N1, H5N1, H7N9 and H9N2, influenza B Virus, respiratory syncytial virus types A and B, human parainfluenza virus type II, adenovirus types 3 and 7, enterovirus EV71, Mycoplasma pneumoniae, Epstein-Barr virus, human cytomegalovirus, and Mycobacterium tuberculosis were synthesized.The extracted nucleic acids were added to the RHAM reaction mix.There was no cross-reactivity with other respiratory pathogens, with only SARS-CoV-2 displaying fluorescent signals at a run time of up to 40 min (Fig. 4A,B), thus reflecting the specificity of the RHAM system.These data prove that RHAM is a rapid, sensitive, and specific technology for nucleic acid detection.

Fast and accurate detection of SARS-CoV-2 from clinical samples by RHAM without RNA extraction
To assess the ability of RHAM in POCT, the detection of clinical nasopharyngeal samples without RNA extraction was performed.Nasopharyngeal swabs were collected from patients and added to the storage medium.Positive and negative samples were first determined to use commercially available RT-PCR kits.To evaluate the performance of RHAM, 10 SARS-CoV-2 positive and 10 SARS-CoV-2 negative samples were included and randomized.For RHAM detection, 5 μL of the medium was mixed with 5 μL of lysis buffer.The mixture was then added to the RHAM reaction mix to a final total volume of 25 μL (Fig. 5A).Positive results were obtained for all RT-PCR confirmed positive samples using the RHAM platform within 25 min (Fig. 5B).Moreover, all the SARS-CoV-2 negative samples did not demonstrate any fluorescence signals at a run time of up to 40 min (Fig. 5B).And among the test of 100 clinical samples, RHAM showed 100% agreement with RT-PCR in both positive and negative samples (Fig. 5C,D and Table 1).The data demonstrate 100% accuracy in SARS-CoV-2  A single-stranded DNA (ssDNA) complementary to the probe is synthesized to evaluate the cleavage ability of RNase HII.RNase HII and probe were incubated at 65 °C with (blue) and without (green) ssDNA.(E) Combination of LAMP and RNase HII reporting system.RNase HII and the probe targeting Orf1ab were added to the LAMP reaction mixture.With SARS-CoV-2 RNA (blue); no template control (green).(F) Combination of LAMP and RNase HII reporting system.RNase HII and the probe targeting N gene were added to the LAMP reaction mixture.With SARS-CoV-2 RNA (blue); no template control (green).copies/mL (red), 5 × 10 4 copies/mL (yellow), 5 × 10 3 copies/mL (blue), 5 × 10 2 copies/mL (purple), and no template control (NTC, green).(C) The sensitivity of LAMP was evaluated by adding 10 μL of SARS-CoV-2 nucleic acid with different concentrations to the LAMP reaction mixture targeting Orf1ab (25 μL reaction volume). 1 × 10 5 copies/ mL (red), 5 × 10 4 copies/mL (yellow), 5 × 10 3 copies/mL (blue), 5 × 10 2 copies/mL (purple), and no template control (NTC, green).(D) The sensitivity of LAMP was evaluated by adding 10 μL of SARS-CoV-2 nucleic acid with different concentrations to the LAMP reaction mixture targeting N gene (25 μL reaction volume).5 × 10 5 copies/mL (red), 5 × 10 4 copies/mL (yellow), 5 × 10 3 copies/mL (blue), 5 × 10 2 copies/mL (purple), and no template control (NTC, green).
(E) the correlation between TTP and copy number of SARS-CoV-2 in RHAM amplification targeting Orf1ab.(F) the correlation between TTP and copy number of SARS-CoV-2 in RHAM amplification targeting N gene.

Discussion
Since the twentieth century, the global outbreak of respiratory pathogens, such as influenza A virus and SARS-CoV-2, highlight the critical need for a fast and reliable diagnostic tool to identify infectious agents, which play a key role in controlling epidemics.Given its rapid and easy-to-use technique, RAT is applied largely in COVID-19 POCT as well as in the self-testing setting.However, the low sensitivity at disease onset hinders RAT from becoming an ideal disease-control tool 12,13 .As nucleic acid amplification technology (NAAT) is analogous to PCR, isothermal amplification technology can amplify nucleic acids of the targeted pathogen with specific primers and has a theoretically higher sensitivity than rapid antigen test (RAT) 17,18 .Since the amplification process is at a constant temperature, the reaction time is much shorter than that of PCR.Combining these advantages, isothermal amplification becomes a promising candidate to develop a disease-control tool.
Current isothermal nucleic acid amplification techniques, such as nucleic acid sequence-based amplification (NASBA), exponential strand displacement amplification (E-SDA), exponential rolling circle amplification (E-RCA), LAMP, helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), and exponential amplification reactions (EXPAR) [19][20][21][22][23][24] , provide rapid and simple experimental procedures for nucleic acid-based diagnosis.Yet, these technologies still have several shortcomings.LAMP and EXPAR might have a risk of non-specific amplification, and it may take efforts to comprehensively eliminate false-positive amplicons caused by the accumulation of non-specific products 9 .The RPA system comprises a complex enzyme mix, and it is difficult to couple crude samples to the RPA system.RCA requires a circular template, often requires an additional ligation step, and its application is complicated.We examined the performance of RHAM and LAMP (Fig. 2), with RHAM demonstrating rapid and accurate detection of RNA samples, reflecting a promising platform for isothermal detection and POCT application.
In summary, we here demonstrate the development of a new isothermal amplification technology termed RHAM, which is based on RNase HII and LAMP, to detect targets rapidly and accurately.Further, with less complex sample preparation methods, RHAM can detect clinical samples as fast as 20 min, demonstrating its tremendous potential to develop a diagnostic tool in POC or even self-testing areas.However, further clinical samples should be analyzed to additionally demonstrate the sensitivity and specificity of the RHAM system.

Figure 1 .
Figure 1.Schematic overview of the RHAM technology.(A) Schematic overview of the fluorescence-based LAMP technology.(B) Schematic diagram showing the targeted sequence and primer designed against Orf1ab.(C) Principle of RHAM.In RHAM, LAMP is coupled with the RNase HII reporting system for signal readout in a single tube.Aided by a LAMP primer set comprising six primers, the target sequence is reverse transcribed and exponentially amplified by Bst DNA polymerase.During the exponential amplification, because of the strand displacement ability of Bst DNA polymerase, part of the amplification product turns into a singlestranded state.The ribonucleotide containing fluorescent probe, labeled with a fluorophore and quencher, can hybridize to the single-stranded amplification product and be cleaved by RNase HII, releasing the fluorophore from the quencher and emitting the fluorescent signal.The remaining single-stranded product can hybrid with the new probe and trigger a new cycle of signal amplification.

Figure 2 .
Figure 2. Development of the RHAM system for robust SARS-CoV-2 detection.(A) Example of fluorescencebased LAMP targeting Orf1ab with SARS-CoV-2 RNA (blue) and no template control (green).(B) Example of fluorescence-based LAMP targeting N gene with SARS-CoV-2 RNA (blue) and no template control (green).(C) Detecting Orf1ab single-stranded DNA with RNase HII.A single-stranded DNA (ssDNA) complementary to the probe is synthesized to evaluate the cleavage ability of RNase HII.RNase HII and probe were incubated at 65 °C with (blue) and without (green) ssDNA.(D) Detecting N gene single-stranded DNA with RNase HII.A single-stranded DNA (ssDNA) complementary to the probe is synthesized to evaluate the cleavage ability of RNase HII.RNase HII and probe were incubated at 65 °C with (blue) and without (green) ssDNA.(E) Combination of LAMP and RNase HII reporting system.RNase HII and the probe targeting Orf1ab were added to the LAMP reaction mixture.With SARS-CoV-2 RNA (blue); no template control (green).(F) Combination of LAMP and RNase HII reporting system.RNase HII and the probe targeting N gene were added to the LAMP reaction mixture.With SARS-CoV-2 RNA (blue); no template control (green).

Figure 4 .
Figure 4. Evaluation of RHAM specificity.(A) The specificity of RHAM targeting Orf1ab was evaluated by testing the nucleic acids of other respiratory pathogens, (B) The specificity of RHAM targeting N gene was evaluated by testing the nucleic acids of other respiratory pathogens.All pathogens including coronavirus (HKU1, OC43, NL63, 229E), SARS coronavirus, MERS coronavirus, influenza A H1N1, H5N1, H7N9, and H9N2, influenza B virus, respiratory syncytial virus types A and B, human parainfluenza virus type II, adenovirus types 3 and 7, enterovirus EV71, Mycoplasma pneumoniae, Epstein-Barr virus, human cytomegalovirus, and Mycobacterium tuberculosis.10 μL of nucleic acid was added to the RHAM reaction mixture (25 μL reaction volume).

Figure 5 .
Figure 5. Performance of RHAM in the evaluation of clinical samples.(A) Schematic overview of RHAM detection of SARS-CoV-2 in clinical specimens.(B) Detection of SARS-CoV-2 in human clinical samples using RHAM.Ten SARS-CoV-2 positive and ten negative samples were analyzed.For RHAM detection, 5 μL of the medium was mixed with 5 μL lysis buffer.The lysis product was added to the reaction buffer, with a final reaction mixture of 25 μL.Positive control (blue); No template control (green); positive samples (red).(C) Ct value distribution of RT-qPCR corresponding to positive samples in 100 samples (Some of the genes in the same SARS-CoV-2 sample were NoCt or positive, resulting in a total of 43 positive samples for the same gene).(D) Positive and negative rates of RHAM based on RT-PCR.